Newly synthesized mRNA which is exported from the nucleus is subject to a variety of competing macromolecular interactions. The 5' cap and poly(A) tail are substrates for binding by cap binding protein and poly(A)-binding protein, respectively. The mRNA is also competent for interaction with the translational machinery by formation of the initiation complex, which includes ribosome subunit binding (Hershey, J. W. B., 1991, Ann. Rev. Biochem. 60:717-55). Interactions which are inhibitory to translation can also occur masking the mRNA and creating a nontranslated pool of messages (Curtis, D., et al., 1995, Cell, 81:171-178; Vasalli, J. D., et al., 1989, Genes & Devel., 3:2163-2171; Richter, J. D., 1991, Bioessays, 13(4):179-183). Some mRNAs are transported and localized to specific subcellular sites, which must involve association with a transport machinery while the mRNA is in transit. Localization of mRNA may also involve interaction with immobile components of the cell in order to anchor the mRNA at the correct destination. mRNAs are also subject to degradation which must involve recognition by degradation enzymes (Pelz, S. W., et al., 1992, Curr. Opin. Cell Biol., 14:979-983; Sachs, A., 1993, Cell, 74(3):413-421). An mRNA, therefore, represents a substrate for many interactions which determine its subcellular location, concentration, and level of expression. Presumably, there are discrete elements within the mRNA that mediate each of these various interactions.
The first suggestion of spatial localization of specific mRNAs within cells came from studies of myelin and myelin basic protein (MBP) mRNA (Colman, D. R., et al., 1982, J. Cell Biol., 95:598-608). Detection of MBP mRNA in a highly purified myelin fraction was the basis for later research that showed the localization of MBP mRNA to the peripheral myelin membranes of oligodendrocytes in vivo (Kristensson, K., et al., 1986, Nature (Lond.), 322:544-547; Verity, N. A., et al., 1988, J. Neurosci. Res., 21:238-248) and in vitro (Holmes, E., et al., 1988, J. Neurosci. Res., 19:389-396; Shiota, C., et al., 1989, Dev. Brain Res., 45:83-94; Barbarese, E., 1991, J. Neurosci. Res., 29:271-281). In retrospect, previous work on targeting of mRNA for secretory and membrane proteins to the rough endoplasmic reticulum (ER) by the nascent polypeptide chain also implicitly describes a localization of mRNA to a subcellular site (Blobel, G., et al., 1975, J. Cell Biol., 67:835-851).
Recently, evidence for the localization of mRNAs to subdomains within the rough ER has been reviewed (Okita, T. W., et al., 1994, Trends Cell Biol., 4:91-96). Functional consequences of RNA localization have been demonstrated in Drosophila. Proper localization of Drosophila bicoid and nanos mRNA is required for establishment of the anterior/posterior axis of the embryo (St. Johnston, D., et al., 1992, Cell, 68(2):201-219). Many RNAs are also localized during creation of the dorsal/ventral axis in Xenopus oocytes (Melton, D. A., et al., 1989, In Ciba Foundation Symposium, Cellular Basis of Morphogenesis, 144:16-30). In addition, motile fibroblasts (Singer, R. H., et al., 1989, J. Cell Biol., 108(6):2343-2353), and terminally differentiated neurons localize specific messages (Garner, C. C., et al., 1988, Nature (Lond.), 336:674-677; Bruckenstein, D. A., et al., 1990, Neuron, 5:809-819; Kleiman, R., et al., 1990, Neuron, 5:821-830). The localization of mRNAs has been extensively reviewed (Steward, O., et al., 1992, Trends Neurosci, 15(5):180-186; Wilhelm, J. E., et al., 1993, J. Cell Biol., 123(2):269-274; St. Johnston, D., 1995, Cell, 81:161-170).
All the currently described cis-acting signals for RNA localization reside in the 3'UTR of the mRNA. Cis-acting signals have been defined for the localization of bicoid (Macdonald, P. M., et al., 1988, Nature (Lond.), 336:595-598; Macdonald, P. M., et al., 1993, Development, 118:1233-1243), nanos (Wharton, R. P., et al., 1991, Cell, 67:955-967; Gavis, E. R., et al., 1992, Cell, 71:301-313), oskar (Ephrussi, A. L., et al., 1992, Nature (Lond.), 358:387-392; Kim-Ha, J., et al., 1993, Development, 119(1):169-178), Vg1 (Mowry, K. L., et al., 1992, Science (Wash. D.C.), 255(5047):991-994), .beta.-actin (Kislauskis, E. H., et al., 1994, J. Cell Biol., 127-1441-451), cyclin B (Dalby, B., et al., 1993, EMBO (Eur. Mol. Biol. Organ.) J., 12(3):1219-1227), K10 (Cheung, H. K., et al., 1992, Development, 114(3):653-661), and even-skipped (Davis, I., et al., 1991, Cell, 67:927-940) mRNAs. There are two examples of mRNA localization in Drosophila where multiple RNA elements have been described that control different steps in a multi-step pathway. The localization of oskar to the posterior pole of Drosphila oocytes occurs in several steps (Kim-Ha, J., et al., 1993, Development, 119(1):169-178). The oskar mRNA first moves from the nurse cells into the oocyte and accumulates at the anterior margin. The final step in oskar mRNA localization is movement to the posterior pole. Recently, the previously defined bicoid element (Macdonald, P. M., et al., 1988, Nature (Lond.), 336:595-598) has been subdivided into independent elements which control different steps in the localization pathway (Macdonald, P. M., et al., 1993, Development, 118:1233-1243).
Previous work has shown that MBP mRNA microinjected into oligodendrocytes is assembled into RNA granules that are transported along the processes and localized to the myelin compartment while control mRNAs (globin and actin) are assembled into RNA granules that remain in the perikaryon (Ainger et al., 1993). The different distribution of these mRNAs is presumably controlled by some aspect of their structure.
A multi-step pathway for MBP mRNA localization in oligodendrocytes has been proposed (Ainger, K., et al., 1993, J. Cell Biol., 123(2):431-441). The pathway includes: assembly of the RNA into granules in the perikaryon, anterograde transport along cellular processes and localization within the myelin compartment. Granule assembly, transport, and localization occur in spatially distinct subcellular compartments (the perikaryon, processes, and myelin compartment, respectively). Specific nucleotide sequences responsible for localization and transport remained unknown prior to the present invention.
The present invention provides sequences isolated from the 3'UTR of MBP which direct transport, localization and increase translational efficiency of native (i.e., MBP) and heterologous mRNA transcripts when present in such transcripts.